DNA in all known cells and viruses is synthesized from deoxynucleoside triphosphates (dNTPs), the precursor substrates that are condensed by DNA polymerase enzymes, releasing pyrophosphate as co-product. No exception to this biosynthetic scheme was ever encountered in nature, whether nucleoside triphosphates are condensed in response to a DNA template acting as co-catalyst, an RNA template or no template at all. The pyrimidine and purine base moieties attached to the common triphosphodeoxyribosyl moiety of dNTPs are not universally conserved in nature. In addition to the four canonical dNTPs, four noncanonical dNTPs (also called “analogs”) bearing an exotic pyrimidine and one bearing an exotic purine were found to be condensed by bacterial viruses (Warren R. A., Annu. Rev. Microbiol. 1980).
The demand for purified deoxyribonucleotides is high for example for DNA synthesis (PCR, DNA chip, etc.) and for reverse transcription in vitro in academic research and medical diagnosis. It extends to numerous nucleoside/nucleotide analogues that are used for in vitro DNA mutagenesis, for DNA labeling, or studying nucleotide metabolic enzymes. In parallel, these modified precursors are also used as antibiotics, antiviral and anticancer agents. The diversification of the “bio-compatible nucleotides” is also a field in expansion with the development of synthetic biology as illustrated by the development of new functional base pairs (Khakshoor O. and Kool E. T., Chem. Commun. 2011) or the chemical synthesis and assembly of genomes (Gibson et al., Science 2010; Gibson et al, Nat. Methods, 2010).
Methods for chemical synthesis of nucleotides are evolving but nucleotides are still difficult to make, isolate and characterize (Burgess. K. and Cook D. Chem. Rev. 2000). Enzyme-mediated syntheses of natural nucleosides and of some analogs (Burgess. K. and Cook D. Chem. Rev. 2000; Mikhailopulo I. A. and Miroshnikov A. I., Mendeleev Commun 2011) have been developed using whole cells or coupled enzymes but the available repertoire of enzymes is still limited.
In fact, the enzymatic synthesis of pure 2′-deoxyribonucleotides and the metabolic engineering for producing such compounds is hindered by the intricacy of biosynthesis and salvage pathways, upstream and downstream of the DNA polymerization step. Each nucleobase (A, C, G, T) is naturally processed separately by highly discriminating enzymes that phosphorylate nucleoside monophosphates into diphosphates. This first step of deoxyribonucleoside phosphorylation is highly specific and, in human, four kinases are required: TK1, TK2, dGK and dCK (Eriksson S. et al, Cell Mol Life Sci 2002). Furthermore, an enzyme, the nucleotide reductase, connects RNA to DNA precursor biosynthesis by converting the ribose into the deoxyribose moiety (rNDP into dNDP or rNTP into dNTP, depending on nucleotide reductase family) through a cumbersome and fragile free-radical mechanism (Nordlund P. and Reichard P., Annu. Rev. Biochem. 2006).
Moreover, chemical phosphorylation requires several steps of protection, deprotection and purification (Johnson D. C., et al, Curr. Protoc. Nucleic Acid Chem, 2004). It is only recently that a one-pot synthesis of deoxyribonucleoside 5′-triphosphates without any protection on the nucleosides was reported (Caton-Williams J. et al, Org. lett. 2011). Enzymatic methods using deoxyribonucleosides 5′-monophosphate (dNMP) have been attempted but they require dNMP kinases and pyruvate kinases (Bao J. et al, Biotechnol. 2007; Ladner W. E. et al, J. Org. Chem. 1985).
This complex mechanism renders in vitro dNTPs synthesis very difficult, as it necessitates the use of several enzymes, each of them having only specific substrates and conducting tightly controlled reactions.
There is therefore an urgent need to identify new mechanisms of nucleoside synthesis, e.g., new enzymes that can be more easily reduced to practice, so as to generate new (or common) nucleosides of interest and thereby expand in vitro synthetic DNA chemistry. In particular, as the first limiting step of generating dNTPs or analogs thereof is deoxynucleoside phosphorylation, there is an urgent need to identify enzymes that are able to efficiently exchange deoxyribose mono-, di-, or tri-phosphate between any kind of nucleobases.
In this context, the present inventors identified new enzymes that are able to transfer 5′-phosphorylated deoxyribose between two nucleobases (N, N′), according to the following formula I:dN−(P)x+N′⇄N+dN′−(P)x where ‘x’ stands for 0, 1, 2 or 3. These enzymes are not substrate-specific as they are able to transfer said deoxyribose moiety between any nucleobase (A, T, G, or C), would it be canonical or not (analogs). Moreover, these enzymes have advantageous mono-, di-, and triphosphodeoxyribosyltransferase activities. They are therefore very promising tools for synthesizing various dNTPs or analogs thereof. The use of these enzymes therefore opens the road to new synthetic pathways of deoxyribonucleotides.